Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

Caroline Gamerith,Michael T Zumstein,Sabine Zitzenbacher,Georg M Guebitz,Marco Vastano,Enrique Herrero Acero,Sahar M Ghorbanpour,Michael Sander,Alessandro Pellis,Doris Ribitsch

doi:10.3389/fmicb.2017.00938

Abstract

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.

Highlights

Plastic materials are ubiquitous in our daily life and the annual European production is in a steady state since a decade, the global production is constantly increasing1
No significant differences between the hydrolysis efficiency of the two glycosylation site knock out mutants could be observed, but treatment with the knock out mutants resulted in slightly increased terephthalic acid (TPA) levels compared to the native Thc_Cut1
It is interesting to note that while the two glycosylation site knock out mutants showed very similar results with regards to both expression as well as poly(ethylene terephthalate) (PET) hydrolysis rates, the purification yields of purified Thc_Cut1_ko_Asn were significantly lower compared to Thc_Cut1_ko_ST

Summary

Introduction

Plastic materials are ubiquitous in our daily life and the annual European production is in a steady state since a decade, the global production is constantly increasing1 Most conventional plastics such as polyethylene, polypropylene, polystyrene, poly(vinyl chloride), and poly(ethylene terephthalate) (PET) are fully petrol-based and not biodegradable. Recent innovations allow the biotechnological production of bio-based monomers from renewable carbon, enabling the replacement of petrochemical building blocks (Pellis et al, 2016c,d) These bio-based building blocks can be either produced by microbial conversions of various feedstocks or with combined biotechnological-chemical pathways that lead to various monomers such as 1,4-butanediol and adipic acid (used for the production of PBAT) (Harmsen et al, 2014). The monomers for PEF (2,5-furandicarboxylic acid and ethylene glycol) can be 100% produced from renewable feedstocks (Pellis et al, 2016d)

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